1. Field of Use
This application relates to methods of culturing bone marrow cells such that they express neuronal phenotype for use in transplantation.
2. Background Information
Neurobiologists have long considered the neurons in the adult brain to be like a precious nest egg: a legacy that dwindles with time and illness and is difficult if not impossible to rebuild. Parkinson""s and Alzheimer""s are examples of neurodegenerative diseases whose cures await scientists overcoming the difficulty of rebuilding neurons in the human adult brain. Parkinson""s disease (PD), is a disorder of middle or late life, with very gradual progression and a prolonged course. HARRISON""S PRINCIPLES OF INTERNAL MEDICINE, Vol. 2, 23d ed., Ed. by Isselbacher, Braunwald, Wilson, Martin, Fauci and Kasper, McGraw-Hill Inc., New York City, 1994, pg. 2275. The most regularly observed changes have been in the aggregates of melanin-containing nerve cells in the brainstem (substantia nigra, locus coeruleus), where there are varying degrees of nerve cell loss with reactive gliosis (most pronounced in the substantia nigra) along with distinctive eosinophilic intracytoplasmic inclusions. (Id. at 2276).
In its fully developed form, PD is easily recognized. The stooped posture, the stiffness and slowness of movement, the fixity of facial expression, the rhythmic tremor of the limbs, which subsides on active willed movement or complete relaxation, are familiar to every clinician. Generally, accompanying the other characteristics of the fully developed disorder is the festinating gait, whereby the patient, prevented by the abnormality of postural tone from making the appropriate reflex adjustments required for effective walking, progresses with quick shuffling steps at an accelerating pace as if to catch up with the body""s center of gravity. (Id. at 2276).
Although the modem treatment of PD is more successful than any that was available before the introduction of levodopa, including stereotactic surgery, there are still many problems. (Id. at 2277). Underlying much of the difficulty undoubtedly is the fact that none of these therapeutic measures has an effect on the underlying disease process, which consists of neuronal degeneration. Ultimately, a point seems to be reached where pharmacology can no longer compensate for the loss of basal ganglia dopamine. (Id.).
Alzheimer""s Disease (AD) is due to a degenerative process characterized by progressive loss of cells from the basal forebrain, cerebral cortex and other brain areas. Acetylcholine-transmitting neurons and their target nerves are particularly affected. Senile plagues and neurofibrillary tangles are present. Pick""s disease has a similar clinical picture to Alzheimer""s disease but a somewhat slower clinical course and circumscribed atrophy, mainly affecting the frontal and temporal lobes. One animal model for Alzheimer""s disease and other dementias displays hereditary tendency toward the formation of such plaques. It is thought that if a drug has an effect in the model, it also may be beneficial in at least some forms of Alzheimer""s and Pick""s diseases. At present there are palliative treatments but no means to restore function.
A group of degenerative disorders characterized by progressive ataxia due to degeneration of the cerebellum, brainstem, spinal cord and peripheral nerves, and occasionally the basal ganglia. Many of these syndromes are hereditary; other occur sporadically. The spinocerebellar degenerations are logically placed in three groups: predominantly spinal ataxias, cerebellar ataxias and multiple-system degenerations. To date there are no treatments. Friedrich""s ataxia is the prototypical spinal ataxia whose inheritance is autosomal recessive. The responsible gene has been found on Chromosome 9. Symptoms begin between ages of 5 and 15 with unsteady gait, followed by upper extremity ataxia and dysarthria. Patients are areflexic and lose large-fiber sensory modalities (vibration and position sense). Two other diseases have similar symptoms: Bassen-Kornzweig syndrome (abeta-lipoproteinemia and vitamin E deficiency) and Refsom""s disease (phytanic acid storage disease). Cerebellar cortical degenerations generally occur between ages 30 and 50. Clinically only signs of cerebellar dysfunction can be detected, with pathologic changes restricted to the cerebellum and occasionally the inferior olives. Inherited and sporadic cases have been reported. Similar degeneration may also be associated with chronic alchoholism.
In multiple-system degenerations, ataxia occurs in young to middle adult life in varying combinations with spasticity and extrapyramidal, sensory, lower motor neuron and autonomic dysfunction. In some families, there may also be optic atrophy, retinitis pigmentosa, opthalmoplegia and dementia.
Another form of cerebellar degeneration is paraneoplastic cerebellar degeneration that occurs with certain cancers, such as oat cell lung cancer, breast cancer and ovarian cancer. In some cases, the ataxia may precede the discovery of the cancer by weeks to years. Purkinje cells are permanently lost, resulting in ataxia. Even if the patient is permanently cured of the cancer, their ability to function may be profoundly disabled by the loss of Purkinje cells. There is no specific treatment.
Strokes also result in neuronal degeneration and loss of functional synapses. Currently there is no repair, and only palliation and rehabilitation are undertaken.
Neurotransplantation has been used to explore the development of the central nervous system and for repair of diseased tissue in conditions such as Parkinson""s and other neurodegenerative diseases. The experimental replacement of neurons by direct grafting of fetal tissue into the brain has been accomplished in small numbers of patients in several research universities (including our University of South Florida); but so far, the experimental grafting of human fetal neurons has been limited by scarcity of appropriate tissue sources, logistic problems, legal and ethical constraints, and poor survival of grafted neurons in the human host brain.
One method replaces neurons by using marrow stromal cells as stem cells for non-hematopoietic tissues. Marrow stromal cells can be isolated from other cells in marrrow by their tendency to adhere to tissue culture plastic. The cells have many of the characteristics of stem cells for tissues that can roughly be defined as mesenchymal, because they can be differentiated in culture into osteoblasts, chondrocytes, adipocytes, and even myoblasts. Therefore, marrow stromal cells present an intriguing model for examining the differentiation of stem cells. Also, they have several characteristics that make them potentially useful for cell and gene therapy. Prockop, D.J. Science: 26: 71-74 (1997). This population of bone marrow cells (BMSC) have also been used to prepare dendritic cells, (K. Inaba, et al., J. Experimental Med. 176: 1693-1702 (1992)) which, as the name implies, have a morphology which might be confused for neurons. Dendritic cells comprise a system of antigen-presenting cells involved in the initiation of T cell responses. The specific growth factor which stimulates production of dendritic cells has been reported to be granulocyte/macrophage colony-stimulating factor (GM-CSF). K. Inaba, et al., J. Experimental Med. 176: 1693-1702 (1992).
The presence of stem cells for non-hematopoietic cells in bone marrow was first suggested by the observations of the German pathologist Cohnheim 130 years ago. J. Cohnheim, Arch. Path. Anat. Physiol. Klin. Med. 40: 1 (1867). Cohnheim studied wound repair by injecting an insoluble aniline dye into the veins of animals and then looking for the appearance of dye-containing cells in wounds he created at a distal site. He concluded that most, if not all, of the cells appearing in the wounds came from the bloodstream, and, by implication, from bone marrow. The stained cells included not only inflammatory cells but also cells that had a fibroblast-like morphology and were associated with thin fibrils. Therefore, Cohnheim""s work raised the possibility that bone marrow may be the source of fibroblasts that deposit collagen fibers as part of the normal process of wound repair. The source of fibroblasts in wound repair has been examined in more than 40 publications since Cohnheim""s report of 1867. See, for example, R. Ross, N. B. Everett, R. Tyler, J. Cell Biol. 44: 645 (1970); J. K. Moen. J. Exp. Med. 61: 247 (1935); N. L. Petrakis, M. Davis, S. P. Lusia, Blood 17: 109 (1961); S. R. S. Rangan, Exp. Cell Res. 46: 477 (1967); J. M. Davidson, in INFLAMMATION: BASIC PRINCIPLES AND CLINICAL CORRELATES, J. I. Gallin, I. M. Goldstein, R. Snyderman, Eds. (Raven, New York City, ed. 2, 1992, pp. 809-19; R. Bucala, L. A. Spiegel, J. Chesney, N. Hogan, A. Cerami, Mol. Med. 1: 71 (1994). Most of the data suggest that the fibroblasts are of local origin, but a the issue has not been resolved and is still being examined. Prockop, D. J. Science 26: 71-74 (1997)
Although Cohnheim""s thesis has not yet been substantiated, definitive evidence that bone marrow contains cells that can differentiate into fibroblasts as well as other mesenchymal cells has been available since the pioneering work of Friedenstein, beginning in the mid-1970""s. A. J. Friedenstein, U. Gorskaja, N. N. Kulagina, Exp. Hematol. 4: 276 (1976). Friedenstein placed samples of whole bone marrow in plastic culture dishes and poured off the cells that were nonadherent. The most striking feature of the adherent cells was that they had the ability to differentiate into colonies that resembled small deposits of bone or cartilage. Freidenstein""s initial observations were extended by a number of investigators during the 1980s, particularly by Piersma and associates (A. H. Piersma, R. E. Ploemacher, k. G. M. Brockbank, Br. J. HaematoL. 54: 285 (1983); A. H. Piersma et al., Exp. Hematol. 13: 237 (1985)) and Owen and associates. C. R. Howlett et al., Clin. Orthop. Relat. Res. 213: 251 (1986); H. J. Mardon, J. Bee, k. von der Mark, M. E. Owen, Cell Tissue Res. 250: 157 (1987); J. N. Beresford, J. H. Bennett, C. Devlin, P. S. Leboy, M. E. Owen,J. Cell Sci. 102: 341 (1992). These and other studies (M. E. Owen and A. J. Friedenstein, in Cell and Molecular Biology of Vertebrate Hard Tissues, Ciba Foundation Symp. 136, Wiley, Chichester, UK, 1988, pp. 42-60; S. L Cheng et al., Endocrinology 134: 277 (1994); A. I. Caplan, J. Orthop. Res. 9: 641 (1991); D. J. Richard et al., Dev. Biol. 161: 218 (1994). S. Wakitani, T. Saito, and A. J. Caplan (Muscle Nerve 18: 1412 (1995)) demonstrated that MSCs differentiated into myoblasts and myotubes by treatment with 5-azacytidine and amphotericin B (Fungasome, Gibco). D. Phinney (Prockop, D. J. Science. 26: 71-74 (1997)), recently observed that the cells differentiate into myoblasts and myotubes after treatment with amphotericin B (1 xcexcg/ml) alone; A. J. Friedenstein, R. K. Chailakahyan, U. V. Gerasimov, Cell Tissue Kinet. 20: 263 (1987); A. Keating, W. Horsfall, R. G. Hawley, F. Toneguzzo, Exp. Hematol. 18: 99 (1990); B. R. Clark and A. Keating, Ann. N. Y Acad. Sci 770: 70 (1995)) established that the Marrow Stromal Cells (MSCs) isolated by the relatively crude procedure of Friedenstein were multipotential and readily differentiated into osteoblasts, chondroblasts, adipocytes, and even myoblasts.
Even though the multipotential properties of MSCs have been recognized for several decades, there are surprisingly large gaps in our information about the cells themselves. The cells, isolated by their adherence to plastic as described by Friedenstein (A. J. Friedenstein, U. Gorskaja, N. N. Kulagina, Exp. Hematol. 4: 276 (1976)), initially are heterogeneous and difficult to clone. The fraction of the hematopoietic cells is relatively high in initial cultures of mouse marrow but is less than 30% with human marrow (M. E. Owen and A. J. Friedenstein, in Cell and Molecular Biology of Vertebrate Hard Tissues, Ciba Foundation Symp. 136, Wiley, Chichester, UK, 1988, pp. 42-60; S. L Cheng et al., Endocrinology 134: 277 (1994); A. I. Caplan, J. Orthop. Res. 9: 641 (1991); D. J. Richard et al., Dev. Biol. 161: 218 (1994); A. Keating, W. Horsfall, R. G. Hawley, F. Toneguzzo, Exp. Hematol. 18: 99 (1990); B. R. Clark and A. Keating, Ann. N. Y Acad. Sci. 770: 70 (1995)). Most of the readily identifiable hematopoietic cells are lost as the cells are maintained as primary cultures for 2 or 3 weeks. The cultured MSCs synthesize an extracellular matrix that includes interstitial type I collagen, fibronectin, and the type IV collagen and laminin of basement membranes (M. E. Owen and A. J. Friedenstein, in Cell and Molecular Biology of Vertebrate Hard Tissues, Ciba Foundation Symp. 136, Wiley, Chichester, UK, 1988, pp. 42-60; S. L Cheng et al., Endocrinology 134: 277 (1994); A. I. Caplan, J. Orthop. Res. 9: 641 (1991); D. J. Richard et al., Dev. Biol. 161: 218 (1994); A. Keating, W. Horsfall, R. G. Hawley, F. Toneguzzo, Exp. Hematol. 18: 99 (1990); B. R. Clark and A. Keating, Ann. N. Y Acad. Sci. 770: 70 (1995)). A small fraction of the cultured cells synthesize factor VII-associated antigen and therefore are probably endothelial. The cells secrete cytokines, the most important of which appear to be interleukin-7 (IL-7), IL-8, IL-11, and stem cell factor (c-kit ligand). Conditions for differentiating the cells are somewhat species-dependent and are influenced by incompletely defined variables, such as the lot of fetal calf serum used. However, MSCs from mouse, rat, rabbit, and human readily differentiate into colonies of osteoblasts (depositing mineral in the form of hydroxyapatite), chondrocytes (synthesizing cartilage matrix), and adipocytes in response to dexamethasone, 1,25-dihydroxyvitamin D3, or cytokines such as BMP-2 (A. J. Friedenstein, U. Gorskaja, N. N. Kulagina, Exp. Hematol. 4: 276 (1976); 5-11). In response to 5-azacytidine with amphotericin B (Fungasome, Gibco) or amphotericin B alone, S. Wakitani, T. Saito, and A. J. Caplan (Muscle Nerve 18: 1412 (1995)) demonstrated that MSCs differentiated into myoblasts and myotubes by treatment with 5-azacytidine and amphotericin B. D. Phinney (unpublished data) recently observed that the cells differentiate into myoblasts and myotubes after treatment with amphotericin B (1 xcexcg/ml) alone, and they differentiated into myoblasts that fuse into rhythmically beating myotubes.
Most experiments on the differentiation of MSCs have been carried out with cultures of MSCs as described by Friedenstein (A. J. Friedenstein, U. Gorskaja, N. N. Kulagina, Exp. Hematol. 4: 276 (1976)). For example, U.S. Pat. No. 4,714,680 issued Dec. 22, 1987, discloses a method of harvesting marrow from donors. Monoclonal antibodies that recognize a stage-specific antigen or immature human marrow cells are provided. These antibodies are useful in methods of isolating cell suspension from human blood and marrow that can be employed in bone marrow transplantation. Cell suspensions containing human pluripotent lympho-hematopoietic stem cells are also provided, as well as therapeutic methods employing the cell suspensions.
Several groups of investigators since 1990 have attempted to prepare more homogenous populations. For example, U.S. Pat. No. 5,087,570, issued Feb. 11, 1992, discloses how to isolate homogeneous mammalian hematopoietic stem cell compositions. Concentrated hematopoietic stem cell compositions were substantially free of differentiated or dedicated hematopoietic cells. The desired cells are obtained by subtraction of other cells having particular markers. The resulting composition may be used to provide for individual or groups of hematopoietic lineages, to reconstitute stem cells of the host, and to identify an assay for a wide variety of hematopoietic growth factors.
U.S. Pat. No. 5,633,426 issued May 27, 1997, is another example of the differentiation and production of hematopoietic cells. Chimeric immunocompromised mice are given human bone marrow of at least 4 weeks from the time of implantation. The bone marrow assumed the normal population of bone marrow except for erythrocytes. These mice with human bone marrow may be used to study the effect of various agents on the proliferation and differentiation of human hematopoietic cells.
U.S. Pat. No. 5,665,557 issued Sep. 9, 1997, relates to methods of obtaining concentrated hematopoietic stem cells by separating out an enriched fraction of cells expressing the marker CDw109. Methods of obtaining compositions enriched in hematopoietic megakaryocyte progenitor cells are also provided. Compositions enriched for stem cells and populations of cells obtained therefrom are also provided by the invention. Methods of use of the cells are also included.
U.S. Pat. No. 5,753,505 issued on Jun. 5, 1995, is yet another method of differentiation. Primordial tissue is introduced into immunodeficient hosts, where the primordial tissue develops and differentiates. The chimeric host allows for investigation of the processes and development of the xenogeneic tissue, testing for the effects of various agents on the growth and differentiation of the tissue, as well as identification of agents involved with the growth and differentiation.
U.S. Pat. No. 5,753,505 issued May 19, 1998, provides an isolated cellular composition in comprising greater than about 90% mammalian, non-tumor-derived, neuronal progenitor cells which express a neuron-specific marker and which can give rise to progeny which can differentiate into neuronal cells. Also provided are methods of treating neuronal disorders utilizing this cellular composition.
U.S. Pat. No. 5,759,793 issued Aug. 6, 1996, provides a method for both the positive and negative selection of at least one mammalian cell population from a mixture of cell populations utilizing a magnetically stabilized fluidized bed. One application of this method is the separation and purification of hematopoietic cells. Target cell populations include human stem cells.
U.S. Pat. No. 5,789,148 issued Aug. 4, 1998, discloses a kit, composition and method for cell separation. The kit includes a centrifugable container and an organosilanized silica particle-based cell separation suspension suitable for density gradient separation, containing a polylactam and sterilized by treatment with ionizing radiation. The composition includes a silanized silica particle-based suspension for cell separation that contains at least 0.05% of a polylactam, and preferably treated by ionizing radiation. Also disclosed is a method of isolating rare blood cells from a blood cell mixture.
Within the past several years, MSCs have been explored as vehicles for both cell therapy and gene therapy. The cells are relatively easy to isolate from the small aspirates of bone marrow that can be obtained under local anesthesia; they are also relatively easy to expand in culture and to transfect with exogenous genes. Prockop, D. J. Science 26: 71-74 (1997). Therefore, MSCs appear to have several advantages over hematopoietic stem cells (HMCs) for use in gene therapy. The isolation of adequate numbers of HSCs requires large volumes of marrow (1 liter or more), and the cells are difficult to expand in culture. (Prockop, D.J. (ibid.)).
There are several sources for bone marrow tissue, including the patient""s own bone marrow, that of blood relatives or others with MHC matches, bone marrow banks, and umbilical cord blood banks. There are several patents that encompass this source. U.S. Pat. No. 5,476,997 issued May 17, 1994, discloses a method of producing human bone marrow equivalent. A human hematopoietic system is provided in an immunocompromised mammalian host, where the hematopoietic system is functional for extended periods of time. Particularly, human fetal liver tissue and human fetal thymus are introduced into a young immunocompromised mouse at a site supplied with a vascular system, whereby the fetal tissue results in formation of functional human bone marrow tissue.
A source of implantable neurons that is the most ethically controversial, is that of human fetal tissue. U.S. Pat. No. 5,690,927 issued Nov. 25, 1997, also utilizes human fetal tissue. Human fetal neuro-derived cell lines are implanted into host tissues. The methods allow for treatment of a variety of neurological disorders and other diseases. A preferred cell line is SVG.
U.S. Pat. No. 5,753,491 issued May 19, 1998, discloses methods for treating a host by implanting genetically unrelated cells in the host. More particularly, the present invention provides human fetal neuro-derived cell lines, and methods of treating a host by implantation of these immortalized human fetal neuro-derived cells into the host. One source is the mouse, which is included in the U.S. Pat. No. 5,580,777 issued Dec. 3, 1996. This patent features a method for the in vitro production of lines of immortalized neural precursor cells, including cell lines having neuronal and/or glial cell characteristics, comprises the step of infecting neuroepithelium or neural crest cells with a retroviral vector carrying a member of the myc family of oncogenes.
U.S. Pat. No. 5,753,506 issued May 19, 1998, reveals an in vitro procedure by which a homogeneous population of multipotential precursor cells from mammalian embryonic neuroepithelium (CNS stem cells) was expanded up to 109 fold in culture while maintaining their multipotential capacity to differentiate into neurons, oligodendrocytes, and astrocytes. Chemical conditions are presented for expanding a large number of neurons from the stem cells. In addition, four factorsxe2x80x94PDGF, CNTF, LIF, and T3xe2x80x94have been identified which, individually, generate significantly higher proportions of neurons, astrocytes, or oligodendrocytes. These procedures are intended to permit a large-scale preparation of the mammalian CNS stem cells, neurons, astrocytes, and oligodendrocytes. These cells are proposed as an important tool for many cell- and gene-based therapies for neurological disorders.
Another source of stem cells is that of primate embryonic stem cells. U.S. Pat. No. 5,843,780 issued Dec. 1, 1998, utilizes these stem cells. A purified preparation of stem cells is disclosed. This preparation is characterized by the following cell surface markers: SSEA-1 (xe2x88x92); SSEA-3 (+); TRA-1-60 (+); TRA-1-81 (+); and alkaline phosphatase (+). In one embodiment, the cells of the preparation have normal karyotypes and continue to proliferate in an undifferentiated state after continuous culture for eleven months. The embryonic stem cells lines are also described as retaining the ability to form trophoblasts and to differentiate into tissues derived from all three embryonic germ layers (endoderm, mesoderm and ectoderm). A method for isolating a primate embryonic stem cell line is also disclosed in the patent.
In summary, there is substantial evidence in both animal models and human patients that neural transplantation is a scientifically feasible and clinically promising approach to the treatment of neurodegenerative diseases and stroke as well as for repair of traumatic injuries to brain and spinal cord. Nevertheless, alternative cell sources and novel strategies for differentiation are needed to circumvent the numerous ethical and technical constraints that now limit the widespread use of neural transplantation.
This invention provides a novel source of neuronal tissue that can be used for grafting into the patient""s own brain or spinal cord (autografting). This invention thus bypasses the delicate issue of using pooled fetal tissue and also obviates the need for immunosuppression. This invention will also be used for allografting (transplantation of bone marrow-derived neuronal cells from one individual to another) and xenografting (transplantation of bone marrow-derived neuronal cells from one species to another). Simply stated, bone marrow cells can be induced to become neurons in vitro and in vivo. Neither the concept nor the technique has been previously reported or accomplished.
In one embodiment, there are disclosed non-fetal, non-tumorigenic, bone-marrow derived neuronal cells suitable for grafting into a mammal""s brain or spinal cord.
In another embodiment, there is disclosed a novel method for obtaining neuron-like cells from bone marrow, said method comprising the steps of a) obtaining bone marrow cells; b) selecting for bone marrow stromal cells; and c) incubating the stromal cells with a differentiating agent, for a time sufficient to change the cell phenotype to neuronal. The method may include an additional step of separating out hematopoietic stem cells. The method of selecting bone marrow stromal cells may include placing the bone marrow cells and suitable culture medium in a plastic culture medium container, allowing the bone marrow stromal cells to adhere to the plastic, and removing the other cells by replacing the medium. The method also provides for using retinoic acid, growth factors, fetal neuronal cells or a combination thereof as the differentiating agent. The growth factors include BDNF, GDNF and NGF. Retinoic acid is 9-cis retinoic acid, all-transretinoic or a combination thereof.
A method for obtaining neurons for auto-transplant from an individual""s own bone marrow, includes the steps of a) harvesting the bone marrow; b) selecting for bone marrow stromal cells; c) incubating the bone marrow stromal cells in a medium including a mitogen until there are sufficient cells for transplantation; d) incubating the stromal cells of step c) with a differentiating agent for a time sufficient to change the cell phenotype to neuronal and/or glial. The mitogen is EGF, PDGF or a combination thereof. The method may include an additional step of separating out hematopoietic stem cells. The method of selecting bone marrow stromal cells may include placing the bone marrow cells and suitable culture medium in a plastic culture medium container, allowing the bone marrow stromal cells to adhere to the plastic, and removing the other cells by replacing the medium. The differentiating agent is selected from retinoic acid, growth factors, fetal neuronal cells or a combination thereof. The growth factors are BDNF, GDNF and NGF, or a combination thereof. Retinoic acid is 9-cis retinoic acid, all-transretinoic or a combination thereof.
Also disclosed is a cell line of bone marrow stromal cells developed by an above method, such that the cells have the ability to migrate and localize to specific neuroanatomical regions where they differentiate into neuronal cells typical of the region and integrate in characteristic architectonic patterns.
Also disclosed is a kit for neuronal cell transplantation with a syringe suitable for obtaining bone marrow, a plastic flask with dehydrated culture medium; and bone marrow stem cell.
Also disclosed is a method of treating a neurodegenerative disorder including administering a sufficient quantity of the cells produced by an above method to an individual with said neurodegenerative disorder. Specific neurodegenerative disorders include Parkinson""s Disease, Alzheimer""s disease, ischemia, spinal cord damage, ataxia, and alcoholism.
Another embodiment is a method of screening for effects of substances on human neuronal cells. The method calls for providing a mammal with implanted human neuronal cells, said cells having migrated to at least one specific location in the mammal""s brain; administering the mammal the substance; and observing the mammal for an effect of the substance.